Experiment 2 Determination of Vitamin C content in vegetables and fruits Preface: Physiological Functions Vitamin C, namely ascorbic acid, is vital for us: – Electron donor with powerful antioxidant capacity Protect and even re-generate other antioxidants: Vit.E, Vit.A, GSH Facilitate gastrointestinal absorption of iron Inhibit the synthesis of N-nitrosamine from nitrite and nitrate – Play a role in hydroxylation reactions Detoxification Collagen synthesis: triple helix is formed after hydroxylation of proline and lysine (proline/prolyl hydroxylase; lysine/lysyl hydroxylase) Biosynthesis of neurotransmitters: norepinephrine from dopamine (dopamine beta hydroxylase); 5-hydroxi-tryptophan from tryptophan Synthesis of carnitine essential for the transport of fatty acids into mitochondria for ATP generation Promote cholesterol metabolism and excretion – Prevent scurvy: most important function Preface: Properties Easily soluble in water, so easily lose by over-washing or with discarded cooking soup Easily destroyed by oxidation: – Heat or prolonged cooking – Exposure to air or alkaline medium – Contact with copper or iron Determination of vitamin C • Determination of total Vitamin C • Determination of reduced Vitamin C Reduced De-hydrogen Part 1 Determination of reduced Vitamin C content (by 2,6-dichlorophenol indophenol) Principle DCPIP (2,6-dichlorophenol indophenol) is a kind of red dye in possession of oxidizing capacity DCPIP loses red when reduced by reduced ascorbic acid through redox reaction – DCPIP fades when reduced ascorbic acid is excessive – DCPIP displays its original red color once the reduced ascorbic acid is depleted Therefore, the content of reduced ascorbic acid can be calculated according to the consumed DCPIP during titration – End point: light red (over 15 seconds) Amount of consumed dye direct proportion Reduced ascorbic acid content Procedure Demarcation of DCPIP solution Determination of sample Calculation Determining the content of DCPIP Standard ascorbic acid solution Titrated by dye Titrated by 0.001M KIO3 Concentration of ascorbic acid solution Concentration of dye solution The result is: 1ml dye solution T=0.088 0.088mg ascorbic acid 1.2.2 Determining the reduced forms in sample sample 100g of sample cutting, pounding with 100ml 2% oxalic acid homogenate pentanol weighing 10g 50ml cylinder washing beaker Diluting to with beaker with a lid 1% oxalic acid graduation Shaking well 20ml upper clear solution taper bottle white bole color shaking fade Keeping still filtering 20ml 1% oxalic acid blank taper white bole Keeping still bottle shaking 5ml filtrate 5ml filtrate titrating light red, keeping 15sec 1.2.3 calculation T=0.06 The content of reduced ascorbic acid (mg/100g) TTT=0.062mgVC/1ml dye (V1V0)T 100 W 2. Determination of total ascorbic acid content by 2,4-dinitrophenyl hydrazine 2.1 Principle: Reduced oxidating De-hydrogen Vit.C form red osazone 2,4-dinitrophenyl hydrazine dissolving with making colorimetric assay 85%H2SO4 There is a direct proportion between the amount of osazone and total ascorbic acid. total Vit. C 2.2 procedure 2.2.1 Sample extracting: 4g homogenate diluting ,washing with 1% oxalic acid (beaker) volumetric flask (100ml) 1% oxalic acid diluting to graduation filtering 2.2.2 oxidation 25ml filtrate 100ml taper 0.5g carbon active shaking 30sec bottle filtering 10ml oxidated filtrate 10ml 1% oxalic acid solution mixing well 2.2.3 Formation of osazone Getting three tubes and process as follow. number A B C Oxidation filtrate(ml) 10% thioura(drop) 2.0 1 2.0 1 2.0 1 0 0.5 0.5 2,4-dinitrophenyl hydrazine(ml) A: blank tube. B,C: sample tube. Keeping these tubes at 37oC in water bather for 3 hours, take them out and keep at room temperature, add 0.5ml 2,4-dinitrophenyl hydrazine into tube A. 2.2.4 Dissolving of sha each tube adding 2.5ml 85% H2SO4 slowly along the tube wall, cold water bathe, shaking taking out keeping still for 30min 490nm adjusting zero with tube A colorimetric analysis 2.2.5 Formation of standard curve standard ascorbic acid applying solution(100ug/ml) about 25ml 100ml taper bottle adding 0.5g carbon active, shaking 1 min filtering 10ml filtrate diluting with 1% oxalic acid 100ml volumetric flask final conc.: 10ug/ml Get five tubes process as follows: number 0 Standard ascorbic acid solution (10ug/ml)(ml) 0 Content of ascorbic acid in each tube(ug) 0 1 2 3 4 0.5 1.0 1.5 2.0 5 10 15 20 1% oxalic acid (ml) 10% thioura (drop) 2.0 1.5 1.0 0.5 0 1 1 1 1 1 2%2,4-dinitrophenyl hydrazine 0.5 0.5 0.5 0.5 Doing as the same as the above described for the sample. Drawing standard curve with the light density as ordinate, and the content of ascorbic acid as abscissa. 0.5 Light density standard curve 0 calculation 5 10 15 20 content of ascorbic acid(ug) C 1 content of total ascorbic acid in sample(mg/ 100g) 100 W 1000 C:ug of standard ascorbic acid correspond to sample tube. W:g of sample in sample determination liquid Questions: 1. Why do we always use oxalic acid in the whole process? 2. The concentration of oxalic acid on making homogenate is 2%, but in other steps, it is 1%. The concentration is different ,why?